Amides of hyaluronic acid and the derivatives thereof and a process for their preparation

ABSTRACT

An amide of hyaluronic acid or a derivative thereof which comprises at least one repetitive unit of general formula (1).: wherein R═NR 6 R 7 , or alcoholic group of the aliphatic, aromatic, arylaliphatic, cycloaliphatic, heterocyclic series, OH, O—, alcoholic group of hyaluronic acid, amino group of deacylated hyaluronic acid; R 1 , R 2 , R 3 , R 4 ═H, SO 3 —, acyl group derived from a carboxylic acid of the aliphatic, aromatic, arylaliphatic, cycloaliphatic, heterocyclic series, —CO—(CH 2 ) 2 —COOY; Y=negative charge, or H; R 5 ═—CO—CH 3 , H, SO 3 —, acyl group derived from a carboxylic acid of the aliphatic, aromatic, arylaliphatic, cycloaliphatic, heterocyclic series, acylic group of hyaluronic acid; R 6 =is H or a aliphatic, aromatic, arylaliphatic, cycloaliphatic, or heterocyclic group, substituted or unsubstituted; R 7 =is H or an aliphatic, aromatic, arylaliphatic, cycloaliphatic, or heterocyclic group, substituted or unsubstituted; wherein at least one of R or R 5  forms an amide group.

CROSS-REFERENCE

This application is a Continuation of pending U.S. application Ser. No.10/220,853, filed on Sep. 6, 2002, which is the national stageapplication of PCT/IB99/01254, filed on Jul. 6, 1999, which designatedthe United States and which claims priority to Italian ApplicationPD98A000169 filed on Jul. 6, 1998. The entire contents of the aboveapplications are hereby incorporated by reference.

SUBJECT OF THE INVENTION

The present invention is directed to amides of hyaluronic acid andderivatives thereof for the preparation of pharmaceutical formulations,of biomaterials and for the coating of biomedical objects and theprocess for their preparation.

FIELD OF THE INVENTION

Hyaluronic acid is a heteropolysaccharide composed of alternate residuesof D-glucuronic acid and N-acetyl-D-glycosamine. It is astraight-chained polymer the molecular weight of which varies between50,000 and 13,000, 000 Da depending on the source from which it wasobtained and the methods used to obtain it. It is present in nature inpericellular gels, in the fundamental substance of the connective tissueof vertebrate organisms of which it represents one of the maincomponents, in the synovial fluid of the joints, in the vitreous humor,in the human umbilical cord tissues and in rooster combs.

In recent years, numerous types of hyaluronic acid derivatives have beensynthesized to obtain compounds with pharmacological properties, orcompounds that can be processed in various forms of biodegradable andbiocompatible biomaterials for use in various fields of medicine,surgery and tissue engineering.

Among the amide derivatives reported in the state of the art are knownwater-insoluble compositions constituted by mixtures deriving from thereaction between the carboxyl of hyaluronic acid, a nucleophil, such asan aminic compound, and an activating agent (U.S. Pat. No. 5,760,200;U.S. Pat. No. 4,937,270). Such mixtures are mainly used in theprevention of post-surgical adhesions.

U.S. Pat. No. 5,733,891 describes pharmaceutical compositions containingamide derivatives of hyaluronic acid obtained by reaction of itscarboxyls with basic anti-tumour agents. The purpose of these compoundsis to focus the action of the active principle on the diseased tissuesand to limit any harmful effects on the healthy tissues.

Moreover, there are known amides of glycosaminoglycans, such ashyaluronic acid, with photosensitive compounds bound by polyfunctionalcompounds that act as bridges in the formation of amide bonds (U.S. Pat.No. 5,462,976).

Lastly, there is a known process for the preparation of insoluble amidesby the reaction of active esters of hyaluronic acid with amines. (WO95/24429).

The aim of the present invention is to provide isolated andcharacterized amides of hyaluronic acid or derivatives thereof, obtainedby reacting the carboxy groups or amino groups originating fromdeacetylation reactions, with amines and acids of the aliphatic,aromatic, arylaliphatic, cycloaliphatic, heterocyclic seriesrespectively, and without the use of spacer chains.

Said compounds can be either water soluble or insoluble, according tothe acid, the amine, the percentage of amide bond or the derivative ofhyaluronic acid used to prepare the amide.

Therefore, the products according to the present invention are suitablefor a large number of applications according to their solubility inwater, their viscosity and the stability of the amide bond.

Indeed, said compounds can be used to prepare both pharmaceuticalcompositions and biomaterials. Moreover, they have the advantage ofbeing able to be formed by reaction, not only with amines, but also withpharmacologically active acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the IR spectroscopy analysis of the compound of Example 11.

FIG. 2 shows the IR spectroscopy analysis of the compound of Example 13,and shows a peak at 1537 cm-1 due to bending in the NH plane (the amideband) and a peak at about 730 cm-1 due to bending of the CH outside theplane of the aromatic ring.

FIG. 3 shows the IR spectroscopy analysis of the compound of Example 13and shows the difference between the graph relative to the amide andthat of the sodium salt of hyaluronic acid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to amides of hyaluronic acid andderivatives thereof for the preparation of pharmaceutical formulations,biomaterials and for the coating of biomedical objects and the processfor their preparation.

The amides according to the present invention can be represented by thefollowing general formula that represents the repetitive unit of thepolymer:

wherein:

R═NR₆R₇, or alcoholic group of the aliphatic, aromatic, arylaliphatic,cycloaliphatic, heterocyclic series, OH, O—, alcoholic group ofhyaluronic acid, amino group of deacylated hyaluronic acid;

R₁, R₂, R₃, R₄,═H, SO₃₋, acyl group derived from a carboyxylic acid ofthe aliphatic, aromatic, arylaliphatic, cycloaliphatic, heterocyclicseries, —CO—(CH₂)₂—COOY; Y=negative charge, or H;

R₅═—CO—CH₃, H, SO₃₋, acyl group derived from a carboxylic acid of thealiphatic, aromatic, arylaliphatic, cycloaliphatic, heterocyclic series,acylic group of hyaluronic acid;

R₆=is H or a aliphatic, aromatic, arylaliphatic, cycloaliphatic, orheterocyclic group, substituted or unsubstituted;

R₇=is H or a aliphatic, aromatic, arylaliphatic, cycloaliphatic, orheterocyclic group, substituted or unsubstituted; wherein at least oneof R or R₅ forms an amide group.

These are therefore amides obtained by reaction of an amine with a freecarboxyl of hyaluronic acid or a derivative thereof, or by reaction ofan acid with a deacylated amino group of hyaluronic acid or a derivativethereof

Of the hyaluronic acid derivatives that can be used to prepare amidesaccording to the present invention, the following are preferred:

-   -   hyaluronic acid esters wherein a part or all of the carboxy        functions are esterified with alcohols of the aliphatic,        aromatic, arylaliphatic, cycloaliphatic, heterocyclic series (EP        0216453 B1);—autocross-linked esters of hyaluronic acid wherein        a part or all of the carboxy groups are esterified with the        alcoholic functions of the same polysaccharide chain or other        chains (EP 0341745 B1);    -   the cross-linked compounds of hyaluronic acid wherein a part or        all of the carboxy groups are esterified with polyalcohols of        the aliphatic aromatic, arylaliphatic, cycloaliphatic,        heterocyclic series, generating cross-linking by means of spacer        chains (EP 0265116 B 1) ;    -   hemiesters of succinic acid or the heavy metal salts of the        hemiester of succinic acid with hyaluronic acid or with partial        or total esters of hyaluronic acid (WO 96/357207); the        0-sulphatcd derivatives (WO 95/25751) or N-sulphated derivatives        (PCT/EP98/01973).

Of the amides obtained by reaction of an amine on the carboxyl ofhyaluronic acid or of a derivative thereof, of particular interest arethe water-soluble ones.

By amide is meant a group of the formula —CON=.

Aliphatic means acyclic or pertaining to open-chain or branched carboncompounds such as alkanes, alkenes or alkynes. Examples of an aliphaticmoiety include but are not limited to C₁-C₂₀ noncyclic hydrocarbons andtheir isomers such as methyl, ethyl, propyl, isopropyl, n-butyl,tert-butyl, isobutyl, pentyl, isopentyl, neopentyl, tert-pentyl,2methylbucyl, 1,2-dimethylpropyl, hexyl, isohexyl, 1-methylpentyl, 2methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl,2,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl,3,3-dimethylbutyl, 1ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl,1,2,2-trimethylpropyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,tridecyl, tetradecyl, pentadecyl, hexadecyl, cetyl, heptadecyl,octadecyl, nonadecyl, stearyl, etc.

Aromatic means an aryl moiety having one or more unsaturated rings, eachring usually having 5 to 8 members and preferably 5 to 6 members.

Examples of the aromatic moiety include but are not limited to benzyl,toluyl, napthalyl, anthracenyl, phenanthryl, fluorenyl, coronenyl,triphenylenyl, fluoranthenyl, benzofluoranthenyl, benzopyrenyl, andpyrenyl.

Cycloaliphatic pertains to a carbon ring structure, usually having 3 to8 members and preferably 5 to 6 members, that does not contain aresonance structure. Examples of cycloaliphatic groups include but arenot limited to cycloalkanes and cycloolefins such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cycloactyl,cyclohexenyl (tetrahydrobenzenyl), cyclohexylidenyl, andcyclooctadienyl.

The heterocyclic series pertains to dissimilar atoms in a ring. Aheterocyclic group is a heteroaryl group usually having a 3- to8-membered, preferably 5- to 6-membered ring or fused ring containing atleast one hetero atom (such as O, S, N, etc.) and include but are notlimited to thienyl, furanyl, pyranyl, 2H-pyrrolyl, pyrrolyl, imidazolyl,pyrazolyl, pyridyl, pyrazinyl, pyrimidyl, pyridazinyl, isothiazolyl,isoxazolyl, furazanyl, benzothienyl, isobenzofuranyl, chromenyl,indolindinyl, isoindolyl, indolyl, purinyl, quinolidinyl, isoquinolyl,quinolyl, phthalazinyl, quinazolyl, carbazolyl, acridinyl, andphenanthridinyl.

An arylalkyl group is a group having both aromatic and aliphaticsubstituents as defined above. Examples of arylalkyl groups include butare not limited to ethylbenzenyl, isobutylbenzeneyl, benzyl,ethylbenzyl, propylbenzyl, isopropylbenzyl, butylbenzyl, isobutylbenzyl,cyclohexylbenzyl, styrenyl, and biphenyl.

An acyl group is an organic radical derived from an organic acid by theremoval of a hydroxy group. Examples of acyl groups include but are notlimited to formyl, acetyl, proprionayl, butyryl, valeryl, isovaleryl,pivaloyl; aroyl such as benzenesufonyl, benzoyl, toluoyl, and napthoyl;diacyl groups such oxalyl and succinic anhydride; and heteroaroyls suchas furoyl, nicotnoyl, isonicotinoyl, etc.

Such amides can be used to advantage for the preparation ofpharmaceutical compositions, for example in the form of gels, for thetransport and release of drugs or biologically active substances for usein viscoelastic surgery or in ophthalmic surgery.

The amides according to the present invention can be salified with theheavy metals on the free or sulphuric carboxy groups, meaning by heavymetals the elements of the 4^(th), 5^(th) and 6^(th) periods of theperiodical table such as silver, iron, cobalt, copper, zinc, arsenic,strontium, zirconium, antimony, gold, cesium, tungsten, selenium,platinum, ruthenium, bismuth, tin, titanium and mercury. Said salts canbe used in dermatology, in ophthalmology, in dentistry, stomatology,rheumatology, urology, gynaecology, internal surgery, as foodsupplements, anti-oxidating, anti-rheumatic, anti-tumoural,anti-inflammatory, analgesic and anti-ulcer agents.

Moreover, the amide derivatives can be obtained by reaction of carboxylor deacylated nitrogen of hyaluronic acid or a derivative thereof withan amine or with a pharmacologically active acid respectively, or theymay be salified or simply associated with said compounds.

Of the pharmacologically active substances, the following are preferred:

-   -   antibiotics, anti-infective, antimicrobial, antiviral,        cytostatic, cytotoxic, anti-tumoral, anti-inflammatory and wound        healing agents, anesthetic, analgesics, vasoconstrictors,        cholinergic or adrenergic agonists and antagonists,        anti-thrombotic, anti-coagulant, haemostatic, fibrinolytic and        thrombolytic agents) proteins and their fragments, peptides and        polynucleotides.

Hereafter we report some examples of pharmacologically active substancesbelonging to the aforesaid classes of drugs.

-   -   antibiotics: amino glucosides, macrolides, tetracycline,        peptides such as gentamicin, neomycin, streptomycin,        dihydrostreptomycin, kanamycin, amikacin, tobramycin,        spectinomycin, erythromycin, oleandomycin, carbomycin,        spiramycin, oxytetrarycline, rolitetracycline, bacitracin,        polymyxin B, gramicidin, colistin, chloramphenicol, lincomycin,        vancomycin, novobiocin, ristocetin, clindamycin, amphotericin B,        griseofulvin, nostatin and their salts;    -   anti-infective agents: diethylcarbamazine, mebendazole,        sulfamides such as sulfacetamide, sulfadiazine, sulfisoxazole;    -   anti-virals and anti-tumorals: iodoxuridine, adenine, adenine        arabinoside, trifluorothymidine, acyclovir, ethyldeoxyuridine,        bromovinyldeoxyuridine, 5-iodo-5′-amino-2′,5′-dideoxyuridine;    -   steroid anti-inflammatory agents: dexamethasone, hydrocortisone,        prednisolone, fluorometliolone, medrisone and their esters;    -   non-steroid anti-inflammatory agents: indomethacin,        oxyphenbutazone, fluorbiprofene, dichlofenac, ibuprofen;    -   anesthetic: benoxinate, proparacaine, dibucaine, lidocaine,        benzocaine, benzydamine, bupivacaine and their salts;    -   cholinergic agonists: pilocarpine, methacholine,        carbamylcholine, aceclidine, physostigmine, neostigmine,        demecarium and their salts;    -   cholinergic antagonists: atropine and its salts;    -   adrenergic agonists: noradrenalin, adrenalin, naphazoline,        methoxamine and their salts;    -   adrenergic antagonists: propanol timolol, pindolol, bupranolol,        athenolol, methoprolol, oxprenolol, practolol, butoxamine,        sotalol, butadrinc, labctalol and their salts;    -   antibacterials and disinfectants: nitrofurazone, mafenide,        chlorhexidine, the derivatives of 8-hydroxyquinolinc and their        salts ;    -   cytotoxics : fluorouracil, methotrexate, podophyllin.

Of particular interest are the forms for the transport and release ofthe above said substances and of biologically active substances such asproteins and their fragments, peptides, polynucleotides, growth factors,enzymes, vaccines, substances used in the treatment of diseasesassociated with genetic defects such as those depending on enzymatichypo- or hyper-activity due to defects of the gene encoding for a givenenzyme, deforming diseases and hereditary diseases.

The amide derivatives according to the present invention, in associationwith radioactive and non-radioactive substances, used in contrastsystems, can be used as markers in in vivo diagnostics, for theidentification and treatment of tumour tissues or damaged tissues.

One considerable advantage is represented by the possibility ofprocessing the amide compounds and their salts in different forms ofbiomaterials such as sponges, films, membranes, threads, tampons,nonwoven fabric, microspheres, nanospheres, gauzes, gels and guidechannels. Said biomaterials, used in one or more associated forms, maybe constituted by one or more amide derivatives and their salts,optionally in association with other natural, synthetic or semisyntheticpolymers, and optionally, with biologically active substances.

Some examples of natural polymers that can be used are collagen,coprecipitates of collagen and glycosaminoglycans, cellulose,polysaccharides in the form of gels such as chitin, chitosan, pectin orpectic acid, agar, agarose, xanthane, gellan, alginic acid or thealginates, polymannans or polyglycans, starch and natural gums.

Semisynthetic polymers, for example, can be chosen from the groupconsisting of collagen cross-linked with agents such as aldehydes orprecursors of the same, dicarboxylic acids or their halogenides,diamines, derivatives of cellulose, hyaluronic acid, chitin or chitosan,gellan, xanthane, pectin or pectic acid, polyglycans, polymannan, agar,agarose, natural gum or glycosaminoglycans.

Lastly, examples of synthetic polymers that can be used are polylacticacid, polyglycolic acid or copolymers of the same or their derivatives,polydioxanes, polyphosphazenes, polysulphonic resin, polyurethanes,PTFE.

The above said biomaterials can be used to advantage in various fieldsof surgery, such as in internal and osteo-articular surgery,neuro-surgery, anastomotic, viscoelastic, ophthalmic, oncological,plastic and aesthetic, otorhinolaryngological, abdominal and pelvic,urogynaecological, cardiovascular surgery, in the prevention ofpost-surgical adhesions and hypertrophic scarring.

Moreover, the amide compounds in association with fibrin, and optionallyother biologically active substances, can be used for the preparation ofsurgical glues.

The biomaterials according to the present invention can be used not onlyin the field of surgery but also in haemodyalisis, cardiology,dermatology, ophthalmology, otorhinolaryngology, dentistry,orthopaedics, gynaecology, urology, in extra-corporeal blood circulationand oxygenation, in cosmetics and in angiology.

Said biomaterials, in their various forms, can be used to advantage asscaffolds on which to grow cells such as mesenchymal cells or maturecells to obtain connective, glandular and nerve tissue.

These biopolymers can also be used in the processes of coating objectsused both in the medical field and in industrial sectors, giving newbiological characteristics to the surfaces of the material used as asupport.

Examples of the objects that can be coated are : catheters, guidechannels, probes, cardiac valves, soft tissue prostheses, prostheses ofanimal origin such as cardiac valves from pigs, artificial tendons, boneand cardiovascular prostheses, contact lenses, blood oxygenators,artificial kidneys, hearts) pancreas) and livers, blood bags) syringes,surgical instruments, filtration systems, laboratory instruments,containers for cultures and for the regeneration of cells and tissues,supports for peptides, proteins and antibodies.

The process of coating the surface of such objects can be performed, forexample by the

Plasma Coating technique, described in the international patentapplication by the Applicant, publication No. W096/24392.

The process for the preparation of amides on the nitrogen of hyaluronicacid or one of its deacetylated derivatives can be summarised as thefollowing steps:

-   -   deacetylation reaction, for example, by reaction with hydrazine        sulphate (J. Riesenfeld, Analy. Bioch. 1990, vol. 188, pp        383-389); preparation of the quaternary ammonium salt of the        deacetylated compound such as the tetrabutylammonium salt;    -   preparation of the acylating agent in the form of an active        ester, for example, of paranitrophenylester of aliphatic,        aromatic, arylaliphatic, cycloaliphatic or heterocyclic acid,        chosen for the formation of the amide;    -   N-acylation reaction between the quaternary ammonium salt of        hyaluronic acid or of one of its deacetylated derivatives and        the acylating agent.

The compound is analytically characterised by the following methods:analysis of the percentage of free amino groups:

-   -   the method described by J. Riesenfeld (Analy. Bioch. 1990, vol.        188, pp 383-389);    -   mean molecular weight:    -   this is determined by GPC using a set of Shadex B-803 and B-806        columns, and RI and MALLS equipment;    -   IR and UV spectroscopy analysis:    -   TLC analysis.    -   The sample is hydrolysed in a 1 mol. solution of sodium        hydroxide for 2-4 hours at 70° C. and then acidified with a 1        mol. solution of hydrochloric acid. The acid that is released        during hydrolysis is extracted with organic solvent. The dry        organic extract is analysed by HPLC.    -   % of N-acylation (hydrolysis of the amide)    -   two types of analysis are performed to measure the percentage of        N acylated groups :    -   a) the method described by J. Riesenfeld (Analy. Bioch. 1990,        vol. 188, pp 383-389);    -   b) the sample is hydrolysed in a 1 mol. solution of sodium,        hydroxide for 2-4 hours at 70° C. and then acidified with a 1        mol. solution of hydrochloric acid. The acid that is released        during hydrolysis is extracted with organic solvent. The dry        organic extract is analysed by HPLC.

Preparation of the amides on the carboxyl of hyaluronic acid or aderivative thereof consists in activating the carboxy groups by reactionof the same, in acid form or in the form of quaternary ammonium salt,with an agent such as carbonyldiimidazole, which converts carboxylicacid in the reactive form of an acylating agent.

Said reaction can be performed by catalysis with hydrochloric acid oracid resin and with an amine of the aliphatic, aromatic, arylaliphatic,cycloaliphatic and heterocyclic series.

Characterisation of the compounds includes the following methods:

-   -   IR and UV spectroscopy :    -   Chromatographic analysis.    -   The sample is hydrolysed in a 1 mol. solution of sodium        hydroxide for 2-4 hours at 70° C. and the amine that is released        during hydrolysis is extracted with organic solvent. The dry        organic extract is analysed by HPLC.

The percentage of amidation of the product is generally in the range ofabout 1% to about 90%, more preferably in the range of about 5% to about60%, and most preferably in the range of about 20% to about 50%.

Example 1

Preparation of Partially N-Deacetylated Hyaluronic Acid in the Form ofSodium Salt (DHA/Na)

One gram of sodium hyaluronate, with a mean molecular weight of 600 Kda,is solubilised in 50 ml of a 1% solution of hydrazine sulphate inhydrazine monohydrate.

This is left to react under agitation for five days (120 hours) at 55°C., after which the reaction is stopped by adding 100 ml of ethanol.

The precipitate thus formed is filtered through a Gooch crucible, washedwith ethanol and then dried at room temperature at reduced pressure.

Any hydrazide of hyaluronic acid that will probably be fanned during thereaction with hydrazinolysis is destroyed by reaction with HI0₃ (iodicacid). As the reaction may be very vigorous, it is conducted whilecooling the reaction container in iced water.

The product of hydrasinolysis is solubilised in 50 ml of a solution of5% sodium acetate and reacted with 25 ml of a 0.5 M solution of iodicacid.

The reaction proceeds for 30 minutes under agitation, after which 5 mlof a 57% solution of HI is added to destroy any unreacted HIO₃.

The iodine that has formed is extracted from the aqueous solution withat least three 30-ml aliquots of ethyl ether (until complete decolouringof the aqueous phase). The aqueous solution is brought to neutral pH byadding a solution of NaOH 0.5M followed by treatment with 100 ml ofethanol.

The precipitate obtained is filtered with a Gooch crucible, washed withethanol and then dried at room temperature and at reduced pressure.

The product obtained is characterised analytically to determine thepercentage of N-dcacetylated groups and the mean molecular weight.

Yield of the reaction 90% % of N-deacetylation 26% mean molecular weight130 Kda

Example 2

Preparation of the Salt of Hyaluronic Acid Partially N-Deacetylated withTetrabutylammonium (DHAITBA).

One gram (2.5 mmol.) of hyaluronic acid sodium salt, partiallyN-deacetylated, is solubilised in 60 ml of water and the solution ispercolated through a column filled with 25 ml of a sulphonic resin inthe form of tetrabutylammonium salt (TBA). The sulphonic resin in H formis activated with a 40% solution w/v of TBAOH.

The eluate, containing N-deacetylated hyaluronic acid TBA salt iscollected and freeze-dried.

Example 3

Preparation of p-NO₂-Phenylester of Benzoic Acid (Acylating Agent)

Ten grams (0.082 mol.) of benzoic acid is solubilised in 800 ml ofCH₂Cl₂, after which 11.4 g (0.082 mol.) of p-NO₂-phenol and 16.9 g(0.082 mol) of DCC (Dicyclohexylcarbodiimide) are added. The reactionproceeds for 2 hours, while the solution is boiled and refluxed.

Subsequently, the dicyclohexylurea that forms is filtered and thefiltered product is dried with a rotavapor under reduced pressure. Theproduct thus obtained is purified by repeated crystallisation in ethylacetate. The crystals are filtered and placed to dry at room temperatureat reduced pressure.

The derivative is characterised by TLC analysis (eluent: CH₂Cl₂/ethylacetate 90/10 and Rf=0.77) and by IR and UV spectroscopy.

Yield of the reaction 92%

Example 4

Preparation of p-NO₂-Phenylester of Cinnamic Acid (Acylating Agent)

Twelve grams (0.082 mol.) of cinnamic acid is solubilised in 800 ml ofCH₂Cl₂, after which 11.4 g (0.082 mol.) of p-NO₂-phenol and 16.9 g(0.082 mol) of DCC (Dicyclohexylcarbodiimide) are added. The reactionproceeds for 2 hours during which time the solution is boiled andrefluxed.

Subsequently, the dicyclohexylurea is filtered and the filtered productis dried using a rotavapor at reduced pressure. The product obtained ispurified by repeated crystallisation in ethanol, the crystals arefiltered and left to dry at room temperature and reduced pressure.

The derivative is characterised by TLC analysis (eluent: CH₂Cl₂/ethylacetate 90/10 and Rf=0.77) and by IR and UV spectroscopy.

Yield of the reaction 89%

Example 5

Preparation of p-NO₂-Phenylester of Dodecanoic Acid (Acylating Agent)

Sixteen grams of dodecanoic acid is solubilised in 1 litre of CH₂Cl₂,after which 11.4 g (0.082 mol.) of p-NO₂-phenol and 16.9 g (0.082 mol.)of DCC (Dicyclohexylcarbodiimide) are added. The reaction proceeds for 2hours during which time the solution is boiled and refluxed.

Subsequently, the dicyclohexylurea is filtered and the filtered productis dried using a rotavapor at reduced pressure. The product obtained ispurified by repeated crystallisation in ethyl acetate, the crystals arefiltered and left to dry at room temperature and at reduced pressure.

The derivative is characterised by TLC analysis (eluent: CH₂Cl₂/ethylacetate 90/10 and Rf=0.77) and by IR spectroscopy.

Yield of the reaction 93%

Example 6

Preparation of p-NO₂-Phenylester of Stearic Acid (Acylating Agent)

23.3 grams of stearic acid is solubilised in 1 litre of CH₂Cl₂, afterwhich 11.4 g (0.082 mol.) of p-NO₂-phenol and 16.9 g (0.082 mol.) of DCC(Dicyclohexylcarbodiimide) are added. The reaction proceeds for 2 hoursduring which time the solution is boiled and refluxed.

Subsequently the dicyclohexylurea is filtered and the filtered productis dried using a rotavapor at reduced pressure. The product obtained ispurified by repeated crystallisation in absolute ethanol, the crystalsare filtered and left to dry at room temperature at reduced pressure.

The derivative is characterised by TLC analysis (eluent: CH₂Cl₂/ethylacetate 90/10 and Rf=0.82) and by IR spectroscopy.

Yield of the reaction 87%

Example 7

Preparation pf p-NO₂-Phenylester of o-acetyl Salicylic Acid (AcylatingAgent)

14.7 g of acetylsalicylic acid is solubilised in 1 litre of CH₂Cl₂,after which 11.4 g (0.082 mol.) of p-NO₂-phenol and 16.9 g (0.082 mol.)of DCC (Dicyclohexylcarbodiimide) are added. The reaction proceeds for 2hours during which time the solution is boiled and refluxed.

Subsequently, the dicyclohexylurea that forms is filtered and thefiltered product is dried using a rotavapor at reduced pressure. Theproduct obtained is purified by repeated crystallisation in absoluteethanol, the crystals are filtered and left to dry at room temperatureat reduced pressure.

The derivative is characterised by TLC analysis (eluent: CH₂Cl₂/ethylacetate 90/10 and Rf=0.82) and by IR spectroscopy.

Yield of reaction 80%

Example 8

Preparation of Partially N-Acylated Hyaluronic Acid (with the BenzoicAcid Derivative)

One gram (1.6 mmol.) of DHA/TBA (26% deacetylation) is solubilised in 50ml of DMSO, after which 5 ml of a 10% solution of p-NO₂-phenylester ofbenzoic acid (prepared according to example 3) in DMSO is added. Thereaction proceeds for 24 hours, under agitation at room temperature,after which it is blocked by adding 2. 5 ml of a saturated solution ofNaCl.

This is left to react for 30 minutes and then 100 ml of ethanol isslowly added. The precipitate thus obtained is filtered through a Gooch,washed with ethanol and ethyl ether and lastly dried at room temperatureand at reduced pressure.

The derivative is analysed by TLC (after hydrolysis of the amide),colorimetric analysis of the percentage of free NH₂ groups and IR and UVspectroscopy.

Yield of the reaction 85% % free NH₂ 11% % N-acylation 15%

Example 9

Preparation of Partially N-Acylated Hyaluronic Acid (with the Derivativeof Cinnamic Acid)

One gram (1.6 mmol.) of DHA/TBA (26% deacteylation) is solubilised in 50ml of NMP, after which 10 ml of a 10% solution of p-NO₂-phenylester ofcinnamic acid (prepared according to example 4) in NMP is added. Thereaction proceeds for 24 hours, under agitation, at room temperature,after which it is blocked by adding 2.5 ml of a solution saturated withNaCl. This is left to react for 30 minutes and lastly 100 ml of ethanolis slowly added. The precipitate thus obtained is filtered through aGooch crucible, washed with ethanol/water 9:1, ethyl ether and lastlydried at room temperature at reduced pressure.

The derivative is analysed by TLC (after hydrolysis of the amide),colorimetric analysis of the percentage of free NH₂ groups and IR and UVspectroscopic analysis.

Yield of the reaction 85% % free NH₂ 11% % N-acylation 15%

Example 10

Preparation of Partially N-Acylated Hyaluronic Acid (with a Derivativeof Dodecanoic Acid)

One gram (1.6 mmol.) of DHA/TBA (26% deacetylation) is solubilised in 50ml of NMP, after which 3.2 ml of a 10% solution of p-NO₂-phenylester ofdodecanoic acid (prepared according to example 5) in NMP is added.

The reaction proceeds for 24 hours, under agitation, at roomtemperature, after which it is blocked by adding 2.5 ml of a saturatedsolution of NaCl.

This is left to react for 30 minutes, after which 100 ml of ethanol isgently added. The precipitate obtained is filtered through a Gooch,washed with ethanol and ethyl ether and lastly dried at room temperatureat reduced pressure.

The derivative is analysed by TLC (after hydrolysis of the amide),colorimetric analysis of the percentage of free NH₂ groups and IR and UVspectroscopy.

Yield of the reaction 88% % free NH₂ 10% % N-acylation 16%

Example 11

Preparation of Partially N-Acylated Hyaluronic Acid (with the Derivativeof Stearic Acid)

One gram (1.6 mmol) of DHA/TBA (26% deacetylation) is solubilised in 50ml of NMP, after which 6 ml of a 10% solution of p-NO₂-phenylester ofstearic acid (prepared according to example 6) in NMP is added. Thereaction proceeds for 24 hours under agitation at room temperature afterwhich it is blocked by adding 2.5 ml of a saturated solution of NaCl.This is left to react for 30 minutes and then 100 ml of ethanol isslowly added.

The precipitate thus obtained is filtered through a Gooch filter, washedwith ethanol and ethyl ether and lastly left to dry at room temperatureand reduced pressure.

The derivative is analysed by TLC (after hydrolysis of the amide),colorimetric analysis of the percentage of free NH₂ groups and IR and UVspectroscopy.

Yield of the reaction 85% % free NH₂ 12% % N-acylation 14%

IR spectroscopy (FIG. 1): the figure shows the difference between the IRspectrum of the amide and that of hyaluronic acid sodium salt. In thespectrum of the amide, there is an evident peak in the area of 2900cm-1, due to the stretching of the CH₂ of the stearate.

Example 12

Preparation of Partially N-Acylated Hyaluronic Acid (with AcetylSalicylic Acid Derivative)

One gram (1.6 mmol.) of DHA/TBA is solubilised in 50 ml of NMP, afterwhich 3.2 ml of a 10% solution of p-N02-phenylester of acetyl salicylicacid (prepared according to example 7) in NMP is added. The reactionproceeds for 24 hours under agitation at room temperature, after whichit is blocked by adding 2.5 ml of a saturated solution of NaCl. This isleft to react for 30 minutes and lastly 100 ml of ethanol is slowlyadded. The precipitate thus obtained is filtered through a Goochcrucible, washed with ethanol and ethyl ether and then dried at roomtemperature and reduced pressure.

The derivative is analysed by TLC (after hydrolysis of the amide),colorimetric analysis of the percentage of free NH₂ groups and IR and UVspectroscopy.

Yield of the reaction 90% % free NH₂ 10% % N-acylation 16%

Example 13

Preparation of Benzylamide of Hyaluronic Acid

Two grams (3.2 mmol.) of tetrabutylammonium salt of hyaluronic acid(HA/TBA) is solubilised in 100 ml of DMSO. This solution is supplementedwith 3 ml of humid acid resin in DMSO and 784 mg (4.8 mmol.) of1,1-carbonyldiimidazole. This is left to react under agitation for 12hours, after which it if filtered through a Gooch crucible to eliminatethe resin and the filtered product is supplemented with 1 ml (9.6 mmol)of benzylamine. This is left to react for 48 hours and then 5 ml of asaturated solution of NaCl is added and it is left under agitation for30 minutes. It is supplemented with 200 ml of acetone and theprecipitate thus obtained is filtered and dried at reduced pressure.

The dry derivative is characterised by TLC, IR and HPLC analysis.

% of amidation 25%

IR spectroscopy (FIGS. 2 and 3): the spectrum in FIG. 2 clearly shows apeak at 1537 cm-1 due to bending in the NH plane (the amide band) and apeak at about 730 cm-1 due to bending of the CH outside the plane of thearomatic ring. FIG. 3 shows the difference between the graph relative tothe amide and that of the sodium salt of hyaluronic acid.

Example 14

Preparation of Benzylamide of Hyaluronic Acid

Two grams (3.2 mmol.) of tetrabutylammonium salt of hyaluronic acid(HA/TBA) is solubilised in 100 ml of DMSO. The solution is adjusted topH 3 with HCl 1M and then 784 mg (4.8 mmol.) of 1,1-carbonyldiimidazoleis added. This is left to react under agitation for 12 hours, then it isfiltered through a Gooch crucible to eliminate the resin and 1 ml (9.6mmol.) of benzylamine is added to the filtered product. This is left toreact for 48 hours, then 5 ml of a saturated solution of NaCl is addedand left under agitation for 30 minutes. To this is added 200 ml ofacetone, the precipitate thus obtained is filtered and dried underreduced pressure.

The dry derivative is characterised by TLC, IR and HPLC analysis.

% amidation 15%

Example 15

Preparation of Benzylamide of Hyaluronic Acid

Two grams (5.2 mmol.) of hyaluronic acid in acid form is solubilised in100 ml of DMF. To this solution is added 854 mg (5.2 mmol.) of1,1-carbonyldiimidazole. This is left to react under agitation for 6hours, after which 1.13 ml (10.4 mmol.) of benzylamine is added. Thereaction proceeds for 48 hours, and is then blocked by adding 200 ml ofacetone.

The precipitate thus obtained is filtered and dried under reducedpressure.

The dry derivative is characterised by TLC, IR and HPLC analysis.

% amidation 60%

Example 16

Preparation of Benzylamide of Hyaluronic Acid

Two grams (5.2 mmol.) of hyaluronic acid in acid form is solubilised in100 ml of DMF. To this solution is added 2 ml of pyridine, 3.68 g (0.026mol,) of p-NO₂-phenol and pyridine chloride until a pH of 7/8 isreached.

Lastly, 5.3 (0.026 mol.) of DCC and 2.8 (0.026 mol.) of benzylamine areadded. This is left to react under agitation for 16 hours after whichthe reaction is blocked by adding 200 ml of acetone. The precipitatethus obtained is filtered and dried under reduced pressure.

The dry derivative is characterised by TLC, IR and HPLC analysis.

% amidation 5%

Example 17

Preparation of Benzylamide of Hyaluronic Acid

Two grams (3.2 mmol.) of HA/TBA is solubilised in 100 ml of DMSO. Thesolution is insufflated with gaseous HCl until the reaction mixturereaches a pH of between 4.5 and 5. Subsequently, 518 mg (3.2 mmol.) ofcarbonyldiimidazole is added. It is left to react under agitation forone hour at room temperature, after which 0.700 ml (6.4 mmol.) ofbenzylamine is added. The reaction proceeds for 16-18 hours. After thistime, 5 ml of a solution saturated with NaCl is added. It isprecipitated by adding 200 ml of acetone and the precipitate thusobtained is filtered and dried under reduced pressure.

The dry derivative is characterised by TLC (after hydrolysis), IR andHPLC analysis.

% amidation 50%

Example 18

Preparation of the Octylamide of Hyaluronic Acid

Two grams (3.2 mmol.) of HA/TBA is solubilised in 100 ml of DMSO. Thesolution is insufflated with gaseous HCl till the reaction mixturereaches a pH of between 4.5 and 5. Subsequently, 207 mg (1.28 mmol.) ofcarbonyldiimidazole is added. It is left to react under agitation forone hour at room temperature, after which 0.417 ml (3.2 mmol.) ofoctylamine is added. The reaction proceeds for 16-18 hours. At the endof this time, 5 ml of a solution saturated with NaCl is added. It isprecipitated by adding 200 ml of acetone and the precipitate obtained isfiltered and dried under reduced pressure.

The dry derivative is characterised by TLC (after hydrolysis), IR andHPLC analysis.

% amidation 25%

Example 19

Preparation of the Dodecyl Amide of Hyaluronic Acid:

Two grams (3.2 mmol.) of HA/TBA is solubilised in 100 ml of DMSO. Thesolution is insufflated with gaseous HCl till the reaction mixturereaches a pH of between 4.5 and 5. Subsequently, 104 mg (0.64 mmol.) ofcarbonyldiimidazole is added. It is left to react, under agitation, forone hour at room temperature, after which 600 mg (3.2 mmol.) ofdodecylamine is added. The reaction proceeds for 16-18 hours. After thistime, 5 ml of a solution saturated with NaCl is added. It isprecipitated by adding 200 ml of acetone and the precipitate obtained isfiltered and dried under reduced pressure.

The dry derivative is characterised by TLC (after hydrolysis), IR andHPLC analysis.

% amidation 15%

Example 20

Preparation of the Hexadecylamide of Hyaluronic Acid:

Two grains (3.2 mmol.) of HA/TBA is solubilised in 100 ml of DMSO. Thesolution is insufflated with gaseous HCl till the reaction mixturereaches a pH of between 4.5 and 5. Subsequently, 52 mg (0.32 mmol.) ofcarbonyldiimidazole is added and left to react under agitation for onehour at room temperature, after which 780 mg (3.2 mmol.) ofhexadecylamine is added. The reaction proceeds for 16-18 hours. Afterthis time, 5 ml of a solution saturated with NaCl is added. It isprecipitated by adding 200 ml of acetone and the precipitate obtained isfiltered and dried under reduced pressure.

The dry derivative is characterised by TLC (after hydrolysis), IR andHPLC analysis.

% amidation 5%

The invention being thus described, it is clear that these methods canbe modified in various ways. Such modifications are not to be consideredas divergences from the spirit and purpose of the invention and anymodification that would appear evident to an expert in the field comeswithin the scope of the following claims.

1. A water-soluble amide compound of hyaluronic acid or a derivativethereof which comprises at least one repetitive unit of the followinggeneral formula:

wherein: R═NR₆R₇, R₁, R₂, R₃, R₄═H; R₅═CO—CH₃; R₆=is H; R₇=is aaliphatic, aromatic, arylaliphatic, cycloaliphatic, or heterocyclicgroup, substituted or unsubstituted, with the proviso that R does notform an amide bond with a medicinal agent.
 2. Amidic, water-solublecompounds according to claim 1, obtained by reaction of the carboxylicgroups of hyaluronic acid with an amino group of the aliphatic,aromatic, arylaliphatic, cycloaliphatic, heterocyclic series.
 3. Amidiccompounds according to claim 1, wherein some or any carboxy groups onthe hyaluronic acid derivatives are esterified with aliphatic, aromatic,arylaliphatic, cycloaliphatic, heteroaliphatic alcohols.
 4. Amidiccompounds according to claim 1 or claim 2, salified with heavy metals.5. Amidic compounds according to claim 4, wherein the heavy metals arethose of the 4^(th), 5^(th) and 6^(th) group of the table of elementsand preferably silver, cobalt, iron, copper, zinc, arsenic, strontium,zirconium, antimony, gold, cesium, tungsten, selenium, platinum,ruthenium, bismuth, tin, titanium and mercury.
 6. Amidic compoundsaccording to claim 1 or claim 2, salified with pharmacologically activesubstances.
 7. Amidic compounds according to claim 6, wherein thepharmacologically active substances are antibiotics, anti-infective,antimicrobial, antiviral, cytostatic, antitumoral, anti-inflammatory,wound healing agents, anaesthetics, cholinergic or adrenergic agonistsand antagonists, antithrombotic, anticoagulant, haemostatic,fibrinolytic, thrombolytic agents, proteins and their fragments,peptides, polynucleotides.
 8. A composition comprising at least oneamidic compound and/or salt thereof according to claim 1 or claim 2 andat least one pharmacologically active substance.
 9. A compositionaccording to claim 8, wherein the pharmacologically active substancesare antibiotics, anti-infective, antimicrobial, antiviral, cytostatic,antitumoral, anti-inflammatory, wound healing, anaesthetic agents,cholinergic or adrenergic agonists or antagonists, antithrombotic,anticoagulant, haemostatic, fibrinolytic, thrombolytic agents, proteinsand their fragments, peptides, polynucletotides, growth factors,enzymes, or vaccines.
 10. A composition comprising at least one amidiccompound according to claim 1 or claim 2, and at least one radioactiveor non-radioactive substance.
 11. A biomaterial comprising at least oneamidic compound and/or salt thereof according to claim 1 or claim 2 andat least one other natural, semisynthetic, synthetic polymer. 12.Biomaterials according to claim 11, wherein the natural polymers arecollagen, co-precipitates of collagen and glycosaminoglycans, cellulose,polysaccharides in the form of gels.
 13. Biomaterials according to claim11, wherein the semisynthetic polymers are cross-linked collagen,dicarboxylic acids or their halogenides, diamines, derivatives ofcellulose, hyaluronic acid, chitin or chitosan, gellan, xanthane, pectinor pectic acid, polyglycans, polyrnannan, agar, agarose, natural gum orglycosaminoglycans.
 14. Biomaterials according to claim 11, wherein thesynthetic polymers are polylactic acid, polyglycolic acid or copolymersof the same or their derivatives, polydioxanes, polyphosphazenes,polysulphonic resins, polyurethanes, PTFE.
 15. Biomaterials according toclaim 11, in association with fibrin, and optionally with otherbiologically active substances for the preparation of surgical glues.16. A scaffold for cell cultures comprising the biomaterials accordingto claim
 11. 17. Surgical and health-care articles comprising thebiomaterials according to claim
 11. 18. Biomaterials according to claim11, in the form of guide channels, gauzes, threads, gels, hydrogels,tampons, films, membranes, sponges, non-woven fabrics, microspheres,nanospheres and associations of the same.
 19. Surgical and health-carearticles according to claim 17, in the form of guide channels, gauzes,threads, gels, hydrogels, tampons, films, membranes, sponges, non-wovenfabrics, microspheres, nanospheres.
 20. A biomedical object selectedfrom the group consisting of bypasses, venous catheters, shunts,catheters, guide channels, probes, cardiac valves, artificial tendons,bone and cardiovascular prostheses, contact lenses, soft tissueprostheses, prostheses of animal origin, blood oxygenators, artificialkidneys, hearts, pancreas and livers, blood bags, syringes, surgicalinstruments, filtration systems, laboratory instruments, containers forcell cultures and for the regeneration of cells and tissues, supportsfor peptides, proteins, antibodies, which has been with an amidiccompound of claim 1 or claim
 2. 21. Process for the preparation ofamidic compounds according to claim 1 having the amides on the carboxylof hyaluronic acid or a derivative thereof, involving the followingsteps: a) activation of the carboxy groups of hyaluronic acid orhyaluronic acid derivative by reaction of the same, in the acid form oras a quaternary ammonium salt, with an activating agent, in acidsolution or on acid resin; b) reaction with an amine of an unsubstitutedor substituted aliphatic, aromatic, arylaliphatic, cycloaliphatic,heterocyclic series.
 22. Process according to claim 21, wherein theactivating agent is 1,1-carbonyldiimidazole.
 23. The biomaterialsaccording to claim 13, wherein the cross-linked collagen is cross-linkedwith aldehydes or precursors of the same.
 24. The biomaterial accordingto claim 11, further comprising at least one biologically activesubstance.
 25. A pharmaceutical composition comprising an amide compoundof claim 1 or claim 2 and a pharmaceutically acceptable carrier.
 26. Abiomaterial comprising an amide compound of claim 1 or claim
 2. 27.Biomaterials according to claim 26, in association with fibrin, andoptionally with other biologically active substances for the preparationof surgical glues.
 28. A scaffold for cell cultures comprising thebiomaterials according to claim
 26. 29. Surgical and health-carearticles comprising the biomaterials according to claim
 26. 30.Biomaterials according to claim 26, in the form of guide channels,gauzes, threads, gels, hydrogels, tampons, films, membranes, sponges,non-woven fabrics, microspheres, nanospheres and associations of thesame.
 31. Surgical and health-care articles according to claim 29, inthe form of guide channels, gauzes, threads, gels, hydrogels, tampons,films, membranes, sponges, non-woven fabrics, microspheres, nanospheres.32. The biomaterial according to claim 12, wherein the polysaccharide isselected from the group consisting of chitin, chitosan, pectin or pecticacid, agar, agarose, xanthane, gellan, alginic acid or alginates,polymannans or polyglycans, starch, and natural gums.
 33. Awater-soluble amide compound of hyaluronic acid or a derivative thereofwhich comprises at least one repetitive unit of the following generalformula:

wherein: R═NR₆R₇, R₁, R₂, R₃, R₄═H; R₅═CO—CH₃; R₆=is H; R₇=is aaliphatic, aromatic, arylaliphatic, cycloaliphatic, or heterocyclicgroup, substituted or unsubstituted; with the proviso that R does notform an amide bond with a medicinal agent, and wherein the percentage ofamidation of the compound is up to about 5%.